Genetic information flows from DNA to macromolecular structures; the dominant force in the molecular organization of life. However, virtually all cell surface and secreted proteins in metazoans are modified by the addition of complex carbohydrates in the ER/Golgi secretory pathway. We find that metabolite availability to the Golgi N- glycosylation pathway exerts autonomous control over the assembly of macromolecular complexes on the cell surface, and in this capacity, acts upstream of signaling and gene expression to influence cell growth, differentiation and disease states. GlcNAc branching and number of N-glycans per protein molecule (i.e. occupied N-X-S/T sites) cooperate to regulate binding to galectins and thereby the distribution, clustering and endocytosis of surface glycoproteins in a predictable manner. The number of occupied N-X-S/T sites is an encoded feature of protein sequences, while the extent of GlcNAc- branching is conditional to the enzymatic activity and metabolic supply of UDP-GlcNAc to the medial Golgi N-acetylglucosaminyltransferases I, II, IV and V (Mgat1, 2, 4 and 5). In naive T cells, galectin-3 binds the T cell receptor (TCR) and prevents spontaneous TCR oligomerization in the absence of antigen ligands. This in turn blocks recruitment of Nck, WASp, SLP-76 and CD4-Lck to TCR, F-actin remodeling and transfer of the complex to GM1/cholesterol-enriched microdomains (GEMs). This inhibits basal signaling by the Src tyrosine kinase Lck and TCR clustering and signaling at the immune synapse. The lattice also binds and localizes the tyrosine phosphatase CD45 to GEMs and the immune synapse, thereby suppressing Lck activity and TCR signaling. Once activated, membrane turnover increases and Lck/PI3K/Erk growth signaling stimulates metabolite flux to UDP-GlcNAc biosynthesis and Mgat5 activity, thereby enhancing GlcNAc branching and CTLA-4 surface retention. Mgat5 deficient 129/Sv mice develop late onset spontaneous kidney autoimmunity and are more susceptible to experimental autoimmune encephalomyelitis (EAE), an animal model of MS. EAE susceptibility among inbred mouse strains correlates inversely with GlcNAc branching in T cells. In EAE-susceptible PL/J mice, reduced GlcNAc branching is distributed across deficiencies in at least three Golgi enzymes (Mgat1, 2 and 5). Supplementing T cells with hexosamine pathway metabolites (i.e. GlcNAc, uridine) increases UDP-GlcNAc supply and GlcNAc branching, and in turn suppresses TCR hyperactivity, enhances CTLA-4 surface retention and dampens T cell mediated autoimmunity in PL/J and Non-Obese Diabetic (NOD) mice. In humans, a haplotype of MGAT1 that decreases N-glycan branching in glycoproteins by ~20% synergistically interacts with an allele of CTLA-4 that reduces N-glycan number by ~50%, increasing the risk of Multiple Sclerosis (MS) and Rheumatoid Arthritis (RA) by ~2 fold. The two variants are expected to independently reduce CTLA-4 affinity for galectins and indeed, they act cooperatively to limit CTLA-4 surface levels, a phenotype rescued by metabolic supplementation to UDP-GlcNAc biosynthesis. Taken together our data suggest that genetic and metabolic control of the galectin lattice regulates basal, activation and arrest signaling in T cells, thereby inhibiting autoimmunity in mice and humans. To extend these results, we propose three specific aims. Specific Aim 1 will further characterize critical molecules in T cells regulated by GlcNAc branching. Specific Aim 2 will explore the role of GlcNAc branching in TH17 and Treg differentiation and function. Specific Aim 3 will further define metabolic regulation of GlcNAc branching in T cells. PUBLIC HEALTH RELEVANCE: Autoimmune diseases like Multiple Sclerosis (MS) result from poorly understood interactions between genetic background of the individual and his/her environment. We find that in mice and humans, genetic and metabolic regulation of a pathway that controls the addition of specific sugars to proteins (i.e. protein glycosylation) controls immune hyperactivity and susceptibility to autoimmunity. Metabolically supplementing the pathway with a simple sugar suppresses immune hyperactivity and suppresses autoimmunity in mice and children with treatment-resistant inflammatory bowel disease. Here we further explore the mechanisms by which protein glycosylation regulates immune function and autoimmunity.